Abstract: The invention provides a method that allows the construction of a chimeric and/or modified and/or reconstructed DNA molecule from two DNA fragments in a defined order and orientation, and to clone the molecule one step in a suitable vector using site specific recombination. No initial step of classical cloning via restriction enzymes is needed, in contrast to the classical recombination systems. This method allows the reliability of the recombination method for cloning with the flexibility of PCR to introduce modifications in the insert sequence. Moreover, this method allows the construction of chimerical DNA molecules associating two different elements, such as promoter-gene association or fusion proteins.

Abstract: The present invention relates to the easy cloning of multiple DNA fragments at multiple places in the vector by a single step recombination reaction. More specifically the present invention discloses the use of two recombination sites each having the same pair of recombination sequences and the recombination sites are placed in opposite direction, in order to prevent the recombination between the sites themselves, and offering the opportunity to clone different or the same DNA fragments in multiple sites of the vector. This method is very useful for high throughput cloning of for example a co-suppression vector, or a gene combination vector, or a promoter combination vector or a promoter-gene combination vector, or a gene silencing vector, or a polycistronic RNA vector, or a gene stacking vector, or a biderectional promoter, or combinatorial expression cassettes or a fusion protein.

Abstract: The invention provides a method that allows the construction of a chimeric and/or modified and/or reconstructed DNA molecule from two DNA fragments in a defined order and orientation, and to clone the molecule one step in a suitable vector using site specific recombination. No initial step of classical cloning via restriction enzymes is needed, in contrast to the classical recombination systems. This method allows the reliability of the recombination method for cloning with the flexibility of PCR to introduce modifications in the insert sequence. Moreover, this method allows the construction of chimerical DNA molecules associating two different elements, such as promoter-gene association or fusion proteins.